It is known in the art to use electrochemical electrode sensors for detection in solution of analytes in a solution, including electro-inactive analytes. It is also known to use such sensors for in solution detection of attachment between antibody-antigen pairs, between receptors and ligands, proteins, enzymes, electrocatalysts and metal complexing groups. Conducting electroactive polymers ("CEP's") have shown promise as electrochemical sensor electrodes in such applications. In fabricating such sensor electrodes, a conductive inert metal electrode is treated with a CEP such as polypyrrole, polythiophene, or polyaniline. After fabrication, the CEP becomes the active component of the resultant electrochemical sensor.
For reasons of electrode stability and reproducibility, the conducting polymer coating is applied to the metal electrode by electro-deposition or electropolymerization, often using potentiodynamic, potentostatic, and galvanostatic techniques. Alternatively, a monomer solution in an appropriate solvent can be applied to the metal electrode surface and then evaporated. Because formation of CEP electrodes is known in the art, further details are not presented herein.
FIGS. 1A and 1B depict generic systems that use CEP electrodes for detection, wherein a solution 2 is exposed to a CEP working electrode 4, as well as to a reference electrode 6 and a so-called counter-electrode 8. Generally the reference electrode is coupled to a reference node, preferably ground, and the counter-electrode is electrically coupled to the same node. In FIGS. 1A and 1B, solution 2 is shown as possibly containing target analytes of interest 10 (which analytes may be relatively electro-inactive), and/or other targets 12 that can matingly connect with appropriate receptors 14 affixed to the CEP electrode 4.
The attachment of receptors or ligands 14 to the outer surface of the CEP electrode is known in the art. Receptors 14 that have an attraction affinity targets 12 can include antigens (in which case targets 12 are antibodies), antibodies (in which case targets 12 are antigens), enzymes, proteins, among others.
In the configuration of FIG. 1A, a variable current source 16 is coupled to the CEP electrode 4, and the voltage between the CEP electrode and the reference electrode is measured with a voltmeter (or other apparatus) 18. Typically the current source 16 is slowly varied, and voltmeter reading are recorded. This type of measurement configuration is often referred to as potentiometric.
By contrast, FIG. 1B depicts a so-called amperometric configuration, wherein a variable voltage source 18' is coupled between the CEP electrode and the reference electrode, and current through the CEP electrode is monitored, as with an current meter 16'. Generally, the voltage provided by source 18 is slowly varied or swept (by varying a potentiometer, which is not shown), and current readings are recorded.
CEPs can be electrochemically switched from an oxidized form to a reduced form upon incorporation of a suitable counter-ion during synthesis. When a counter-ion attaches or "hooks" to the CEP, the CEP is said to be doped or oxidized, and when the counter-ion detaches, the CEP is said to be undoped or reduced. By varying the electrical environment to which the CEP is subjected, a neutral, a doped or an undoped state can be made to occur. For example, attachment can occur when a counter-ion is necessary to satisfy a net positive charge on the so-called backbone of the CEP, and detachment can result when there is no longer any need to satisfy the positive net charge on the backbone.
As a CEP electrode incorporates and expels ionic species while switching from an oxidized form to a reduced form, useful analytical signals can be produced. For example, it has long been suggested by Heineman et al. that a CEP may be undoped at a cathodic potential, but redoped when returned to an anodic potential in the presence of an easily incorporated anion analyte, for example phosphate or nitrate. See Y. Ikarayama, C. Caliastsatos, W. R. Heineman, S. Yamanchi, Sens. Act. 12 (1987), 455; Y. Ikarayama, W. R. Heineman, Anal. Chem. 58 (1986) 1803.
Although the precise mechanics of the phenomenon are not completely understood, a counter-ion incorporated during synthesis can have a dramatic effect on the CEP properties, including conductivity, electrochemical switching potential as well as the ion exchange selectivity series.
For example, in FIG. 1A, attachment of an appropriate analyte 10 to the CEP electrode 4 under appropriate bias conditions set by current source 16 can measurably alter the different voltage measured by volt meter 18. By the same token, analyte attachment to the CEP electrode in FIG. 1B under appropriate bias conditions determined by voltage source 18' can alter current flow measured by current meter 16'. Likewise, attraction of suitable targets to receptors 14 in either configuration can also result in a useful analytical signal.
Antigens and antibodies can provide an interaction selectivity, but unfortunately it is difficult in the prior art to generate a meaningful, reproducible analytical signal in response to such interaction. When detecting antigen-antibody attraction, the measurable current or voltage resulting from attraction appears not to be due to any antigen-antibody reaction product. Instead, it is believed that the nature of the CEP itself is changed by the on or off condition of the counter-ion. These problems in attempting to sense a useful interaction signal seem to arise from the lack of a Faradaic (e.g., electron transfer) signal and from the irreversible nature of the antibody-antigen process itself.
The prior art has tried to overcome these measurement problems using potential measurements, indirect amperometric immunoassay techniques, and direct measurements to sense changes in the capacitive nature of the sensor surface after an antibody-antigen interaction. Unfortunately, these procedures are time consuming because of long equilibration times, and/or the multi-step procedures required. Further, the antibody-containing surface must be regenerated chemically to reverse the antibody-antigen interaction.
Antibodies may readily be incorporated into CEPs during synthesis to promote specific reactions with the corresponding antigen. The use of alternating current (AC) voltammetry can provide adequate sensitivity, but reproducibility and the ability to reuse the working electrodes are lacking.
FIG. 1C depicts a cyclic voltammogram ("CV") that is typically produced by the configuration of FIG. 1B, wherein a single sweep is depicted. The data in FIG. 1C is typical of experiments run in a solution of sodium octane sulfonate, wherein the analyte cation will be sodium. Typically voltage source 18' in FIG. 1B is slowly swept at the rate of perhaps 20 mV/second, which means nearly 1.5 minutes are required to sweep 1.6 VDC and generate the data shown in FIG. 1C.
With reference to FIG. 1B and FIG. 1C, the CEP initially is neutral. Let power source 18' initially be about 0 V, whereupon the CEP may be considered to be neutral, or substantially unoxidized. As the voltage sweeps positively (leftwards) to say +0.6 V (e.g., 600 mV), the CEP becomes positively charged (oxidized). To preserve charge balance, this positive charge requires neutralization from negative charges (ions) in the surrounding solution. These ions become incorporated into the CEP structure, and at point B, the current increases as the CEP is fully incorporated (doped) with anions. As these ions migrated into the CEP structure (as a result of the small size of the ions), a discernable current results. Now as the voltage is swept more negatively (e.g., rightwards), the CEP begins to lose the positive charges and is said to be reduced.
To maintain charge neutrality at say about -0.3 V, one of two things can occur. The previously incorporated anions can leave the CEP network, or a cation species from the surrounding solution can be incorporated. As the voltage is made more negative, the CEP becomes further reduced (e.g., less net positive charge). At about -1 V, cations become incorporated, changing the direction and magnitude of current flow. As the voltage is now made more positive (going towards -0.5 V), point A is reached, whereas the CEP is in a reduced state, and begins to become more oxidized once again.
If the voltage sweep were slowly repeated, the current peaks A and B would occur at about the same voltages, but the shape of the CV would probably be changed. The resultant hysteresis would represent fouling and decalibration of the CEP electrode, primarily due to the inability of the polymer to readily de-dope the incorporated charges. Thus, data taken with CEP electrodes at constant potentials are not very reproducible, because targets that incorporate to the CEP tend to remain incorporated, until no further incorporation sites remain. This produces loss of detection sensitivity, and the decalibration and electrode fouling are noted above.
Thus, often after a relatively few minutes of use, the electrode must be replaced or cleansed. It is known to reduce a CEP electrode, e.g., to renew it, by slowly recycling the CEP with an appropriate potential, for example, -1.5 VDC for ten minutes. Understandably, having to replace a CEP electrode or expel targets that have become incorporated into the CEP by electrically reducing the electrode is undesirably time consuming, and disruptive of routine analysis.
In addition to having the working electrode undesirably and apparently irreversibly altered by the experiment, the prior art configurations of FIG. 1A and 1B suffer from other deficiencies. Because these configurations maintain either the current I or the voltage V constant during measurement, no selectivity exists that would allow recognition, for example, between analytes. Because all anions (or cations) may attach to the CEP electrode, one can only measure total anions (or total cations).
As such, non-selective prior art CEP electrodes cannot detect chloride only or fluoride only because all anions interact with the CEP. Even if some anion species interacted differently to the CEP, the prior art cannot discern between the species. For example, prior art applications of CEPs cannot discern between voltage or current change resulting from a relatively low concentration of a very effectively interacting anion species, as contrasted with a larger concentration of a less effectively interacting anion species. The gross detection signals for each could appear identical.
It has also been known in the art to use non-CEP electrodes, e.g., inert metal electrodes that are coupled to a source of periodic voltage. These techniques are often referred to as pulsed coulometric detection ("PCD") or pulsed amperometric detection ("PAD"). PCD methods generally require a chemical-working electrode reaction as an absolute prerequisite to detection.
Typically PCD is an indirect method where, for example, an initial chemical adsorption reaction between the working electrode and hydrogen establishes an electrical current. This current is then attenuated by an adsorption reaction between the working electrode and a chemical analyte. In such applications, although the working electrode may be platinum it cannot, for example, be gold since hydrogen atoms will not be adsorbed by gold. Conversely, regardless of the working electrode composition, the pre-measurement phase electrode-chemical adsorption requirement precludes indirect measurements for those chemicals that do not adsorb to the working electrode.
An improved variation on the PCD technique is described in U.S. Pat. No. 4,939,924 (July 1990) to Johnson et al. wherein a periodic step potential waveform is coupled to an inert metal working electrode, and wherein current integration compensates for measurement noise. Johnson et al.'s method, termed PS-PCD for potential stepped-PCD, more directly detects analyte in a flow-through cell containing a working electrode.
In this method, a pulse-step or ramp-like potential waveform is applied to the working electrode, and the analyte is electrochemically detected directly by integrating current over the cyclic portion of the total potential waveform. This permits Johnson et al. to detect organic molecules based upon measurement of the electrical charge resulting directly from their electrochemical oxidations.
FIG. 1D depicts the improved PCD technique disclosed by Johnson et al. As shown therein, a solution 2 containing target analytes 10 is exposed to an inert metal working electrode 24, as well as to a reference electrode 6 and a counter-electrode 8. A voltage waveform generator 28 is coupled between the working electrode 4 and the reference electrode and outputs a repetitive voltage waveform, such as the waveforms shown in FIGS. 1E, 1F and 1G. A current integrator 30 integrates current in the working electrode to provide a detection signal to a recorder or other instrument 32. As shown in FIGS. 1E-1G, the voltage waveform output by generator 28 typically has a repetition rate of perhaps 60 Hz, and a peak-peak maximum excursion of perhaps one or two volts. The waveform has a first potential value E1, whereat the surface of the working electrode exists at an oxide-free state. The waveform potential then increases from E1 to a higher magnitude E1', to allow an oxide to form on the working electrode surface, with concurrent electrocatalytic oxidative reaction of soluble and/or analyte. The waveform potential returns to the first value E1 for a holding time during which the oxide that formed on the working electrode surface is cathodically stripped off. If the potential is held at E1' sufficiently long, no further oxide reduction is required. Otherwise, the potential may then be elevated to a higher magnitude E2, to accomplish a more thorough oxidative cleaning of the electrode surface. If brought to a negative-most potential E3, electrode reactivation by cathodic dissolution of the surface oxide formed at E1 and/or E2 can occur.
In FIG. 1D, the total time of the detection period is the time at potential E1 plus the time at (or enroute to) potential E1'. Current integrator 30 is activated when potential E1 is first presented, and the integrated current output is sampled after E1', at the end of the return to potential E1.
Unfortunately, an inherent limitation in PCD, PAD, and PS-PCD prior art systems is that electro-inactive analytes cannot be detected. Absent the presence of an electrical charge resulting from an associated electrochemical oxidation, such targets go undetected.
In summary, there is a need for a method and apparatus for detecting targets, including electro-inactive analytes, in a stable, reproducible manner that provides selectivity while inhibiting contamination of the working electrode. Preferably such method and device should find application in flow injection analysis, liquid, and ion chromatography, as well as in capillary electrophoresis.
The present invention discloses such methods and apparatus.